The focus of our research is devoted to understanding the molecular basis for the pathogenesis of retrovirus-induced diseases. Using retroviruses that cause leukemia or neurological disease in rodents, we carry out studies to obtain basic information on how these viruses induce molecular changes in normal cells that result in pathological consequences. We hope to use the information gained from our studies to design rational strategies to counteract the molecular events that are responsible for the diseases induced and apply this to similar diseases in man. To understand the molecular basis for uncontrolled hematopoietic cell growth in a controlled animal model system, we have been studying the erythroleukemia induced in mice by the Friend spleen focus-forming virus (SFFV). Due to expression of gp55, the unique envelope glycoprotein encoded by Friend SFFV, virus-infected erythroid cells can proliferate, differentiate and survive in the absence of erythropoietin (Epo), the normal regulator of erythropoiesis. Our previous studies demonstrated that SFFV gp55, which interacts with the Epo receptor complex at the cell surface, causes constitutive activation of signal transduction pathways normally activated by Epo, including Stat proteins and components of the Ras-MAP kinase and PI 3-kinase/Akt kinase pathways. Our recent studies have focused on understanding how signal transducing molecules become phosphorylated in SFFV-infected cells and on determining the mechanism by which SFFV-infected cells protect themselves against apoptosis after Epo withdrawal. Although our previous studies demonstrated that numerous signal transducing molecules become tyrosine phosphorylated in SFFV-infected cells in the absence of Epo, the mechanism by which this occurs has been unclear. The SFFV envelope protein, gp55, which is responsible for the biological effects of the virus, lacks properties normally associated with signal transducing molecules, such as kinase activity or SH2/SH3 binding domains. Furthermore, SFFV infection does not appear to result in the activation of JAK-2, the major tyrosine kinase involved in Epo signal transduction. When it was recently shown that mice carrying the Fv-2ss gene,which confers susceptibility to SFFV-induced erythroleukemia, express a short form of the receptor tyrosine kinase Stk, called sf-Stk, which is not expressed in resistant mice carrying the Fv-2rr gene, we initiated studies to determine if SFFV gp55 was activating signal transduction pathways indirectly by activating sf-Stk. We demonstrated that SFFV gp55 forms a covalent interaction with this kinase in cells expressing the EpoR, resulting in phosphorylation of sf-Stk and its interaction with the Epo receptor. We recently demonstrated that the interaction of SFFV gp55 and sf-Stk significantly extends the half-life of the kinase. To examine the biological consequences of activated sf-Stk on erythroid cell growth, we prepared a bicistronic vector co-expressing SFFV gp55 and sf-Stk as well as a vector expressing a mutant sf-Stk that is constitutively activated in the absence of SFFV gp55 and tested the effects of these vectors on erythroid colony formation in vitro and their disease potential in mice. Our data indicates that both the SFFV gp55-activated sf-Stk and the constitutively activated mutant of sf-Stk induce erythroid cells from NIH Swiss (Fv-2ss) and C57BL/6 (sf-Stk null,Fv-2rr) mice to form Epo-independent colonies in vitro. Mutational analysis of sf-Stk indicated that a functional kinase domain and 8/12 of its tyrosine residues were required for the induction of Epo-independent colonies in conjunction with SFFV gp55. To determine if activated sf-Stk phosphorylates any of the signal transducing molecules known to be constitutively phosphorylated in SFFV-infected cells, we have been expressing these signal transducing molecules in 293 T cells with gp55-activated sf-Stk. Our data to date indicates that both Erk2 and Gab1 are tyrosine phosphorylated in the presence of SFFV gp55 and sf-Stk and their phosphorylation is dependent upon sf-Stk kinase activity. However, sf-Stk doesn't appear to directly interact with these molecules, suggesting that sf-Stk may be phosphorylating another kinase that subsequently phosphorylates Erk2 and Gab1. We are currently searching for such a kinase. We are also initiating studies to determine if gp55-activated sf-Stk and the constitutively activated mutant of sf-Stk have the same substrates and lead to the phosphorylation of the same signal transducing molecules. In addition to testing the biological effects of gp55-activated and mutation activated sf-Stk on erythroid cells in vitro, we also determined the biological effects of vectors carrying these activated kinases in mice. Although both the bicistronic vector expressing SFFV gp55 and sf-Stk and the constitutively activated sf-Stk mutant induced erythroid cells from Fv-2rr mice to form Epo-independent colonies in vitro and caused erythroleukemia in Fv-2ss mice , neither were able to induce erythroleukemia in Fv-2rrmice, suggesting that these mice carry other genes that block the development of this disease. Interestingly, although injection of NIH Swiss mice with Friend SFFV causes erythroleukemia exclusively, injection of both NIH Swiss and C57BL/6 mice with the bicistronic SFFV gp55-sf-Stk vector resulted in the development of a variety of diseases over a two month period, including hematopoietic neoplasms and hemangiosarcomas. In contrast, injection of mice with the vector expressing the constitutively activated mutant of sf-Stk induced only erythroleukemia and only in NIH Swiss mice. Our studies suggest that SFFV gp55 induces the proliferation and differentiation of erythroid cells in the absence of Epo by covalently interacting with and stabilizing sf-Stk, resulting in the activation of the kinase and various downstream signal transduction pathways. In addition to proliferating and differentiating in the absence of Epo, SFFV-infected erythroid cells, unlike normal erythroid cells, fail to undergo apoptosis when Epo is withdrawn. Our studies indicate that this is due to expression in SFFV-infected cells of high levels of the anti-apoptotic protein Bcl-XL and constitutive phosphorylation of the pro-apoptotic protein BAD, which blocks its activity. Using pharmacological inhibitors, we determined that constitutive expression of Bcl-XL in SFFV-infected cells was dependent upon activation of the MEK/MAP kinase pathway as well as the Stat pathway, and that constitutive phosphorylation of BAD required activation of the PI-3kinase pathway. Studies are in progress to identify the transcription factor(s) regulating the expression of Bcl-XL in Epo-deprived, SFFV-infected cells as well as the kinase responsible for the constitutive phosphorylation of BAD. We recently observed that SFFV infection of erythroid cells leads to a downregulation in the expression of the Fas receptor, which mediates another apoptotic pathway in erythroid cells and this may also contribute to the failure of SFFV-infected cells to undergo apoptosis after Epo. We are currently carrying out studies to determine the mechanism by which Fas is downregulated in SFFV-infected cells. As a second retroviral model system, we have been studying PVC-211 murine leukemia virus (MuLV), a variant of the leukemia-inducing Friend murine leukemia virus that causes a rapidly progressive spongiform neurodegeneration when injected into newborn rats.